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Growth cone turning is an important mechanism for changing the direction of neurite elongation during development of the nervous system. Our previous study indicated that actin filament bundles at the leading margin direct the distal microtubular cytoskeleton as growth cones turn to avoid substratum-bound chondroitin sulfate proteoglycan. Here, we investigated the role of microtubule dynamics in growth cone turning by using low doses of vinblastine and taxol, treatments that reduce dynamic growth and shrinkage of microtubule ends. We used time-lapse phase-contrast videomicroscopy to observe embryonic chick dorsal root ganglion neuronal growth cones as they encountered a border between fibronectin and chondroitin sulfate proteoglycan in the presence and absence of 4 nM vinblastine or 7 nM taxol. Growth cones were fixed and immunocytochemically labeled to identify actin filaments and microtubules containing tyrosinated and detyrosinated ␣-tubulin.Our results show that after contact with substratum-bound chondroitin sulfate proteoglycan, vinblastine-and taxol-treated growth cones did not turn, as did controls; instead, they stopped or sidestepped. Even before drug-treated growth cones contacted a chondroitin sulfate proteoglycan border, they were narrower than controls, and the distal tyrosinated microtubules were less splayed and were closer to the leading edges of the growth cones. We conclude that the splayed dynamic distal ends of microtubules play a key role in the actin filament-mediated steering of growth cone microtubules to produce growth cone turning. Key words: microtubule; growth cone; turning; actin filament; chondroitin sulfate proteoglycan; dynamic instabilityGrowth cones are the motile tips of elongating axons that guide growing axons to their targets during development of the nervous system. Growth cone navigation involves the detection and integration of extracellular signals, followed by a response that can include forward migration, retraction, branching, and turning. Detection of guidance cues is facilitated by protrusion and retraction of filopodia and lamellipodia from the peripheral region (P-domain) of the growth cone, which contains bundles and networks of actin filaments (AFs) (Letourneau and Ressler, 1983;Lewis and Bridgman, 1992). Axonal elongation depends on the advance of microtubules (MTs), which provide structural support and serve as tracks for axonal transport of membranous organelles. Stable MT bundles project from the axon into the central region (C-domain) of the growth cone, whereas dynamic MT ends splay apart and project into the actin-rich P-domain (Letourneau and Ressler, 1983;Gordon-Weeks, 1991;Challacombe et al., 1996).Recent studies indicate that the advance of MTs into specific growth cone regions initiates responses to guidance cues, such as advance toward a target (Lin and Forscher, 1993), turning toward a positive cue (Bentley and O'Connor, 1994), and turning away from an unfavorable substratum or inhibitory guidance cue (Challacombe et al., 1996). Interactions betw...

Sulfated proteoglycans (PGs) may play a significant role in the regulation of neurite outgrowth. They are present in axon-free regions of the developing nervous system and repel elongating neurites in a concentration-dependent manner in vitro. The addition of growth-promoting molecules, such as laminin, can modify the inhibitory effect of PGs on neurite outgrowth (Snow, Steindler, and Silver, 1990b). Substrata containing a high-PG/low-laminin ratio completely inhibit neurite outgrowth, while normal, unimpeded outgrowth is observed on low-PG/high-laminin substrata. Therefore, different patterns of neurite outgrowth may result from regulation of the ratio of growth-promoting molecules to growth-inhibiting molecules. Using video microscopy, embryonic chicken dorsal root ganglia neurons (DRG), chicken retinal ganglia neurons (RGC), and rat forebrain neurons (FB) were analyzed as they extended processes from a substratum consisting of laminin alone onto a step gradient of increasing concentrations of chondroitin sulfate proteoglycan (CS-PG) bound to laminin. In contrast to neurite outgrowth inhibition that occurs at the border of a single stripe of high concentration of CS-PG (Snow et al., 1990b and this study), growth cones grew onto and up CS-PG presented in a step-wise graded distribution. Although the behavior of the different cell types was unique, a common behavior of each cell type was a decrease in the rate of neurite outgrowth with increasing CS-PG concentration. These data suggest that appropriate concentrations of growth-promoting molecules combined with growth-inhibiting molecules may regulate the direction and possibly the timing of neurite outgrowth in vivo. The different responses of different neuronal types suggest that the presence of sulfated PG may have varying effects on different aspects of neuronal development.

The differentiation and morphogenesis of neural tissues involves a diversity of interactions between neural cells and their environment. Many potentially important interactions occur with the extracellular matrix (ECM), a complex association of extracellular glycoproteins organized into aggregates and polymers. In this article, we discuss recent findings on neuronal interactions with the ECM and their roles in neural cell migration and neurite growth. First, we examine the expression and putative functions of the molecules of the neural ECM. Second, we discuss cell surface molecules that mediate neural interactions with ECM components. Last, we address proteoglycans (PGs), a diverse class of glycoproteins, present both as ECM components and as cell surface molecules, which may mediate neural interactions with their environment. The best-understood cellular interactions with the ECM are adhesive, mediated by binding between specific cell surface molecules and cell binding domains of ECM components (Strittmater and Fishman, 199 1; Damsky and Werb, 1992). Cellsubstratum adhesion is necessary for major cell movements of neuron morphogenesis, that is, the migrations of neural cells and their precursors and the migratory behavior ofgrowth cones at the extending tips of axons and dendrites. As cells move, adhesive molecules at the surface of the leading edge of a migrating cell or growth cone bind to ligands on other cell surfaces or ECM components. These bonds stabilize filopodia and lamellipodia, and, in some cases, provide anchorage against which cytoskeletal filaments, associated with the plasma membrane, exert forces to pull the cell or growth cone forward. Thus, ECM has been primarily viewed as an adhesive substratum to provide traction for migrating cells and to stabilize the position and, perhaps, the state of differentiation of nonmotile cells. However, the interactions between neural cells and the ECM are not longer regarded as only adhesive or mechanical. Two points are now clear. First, some of these interactions are definitely not adhesive, but, rather, they may even be antiadhesive (Chiquet-Ehrismann, 199 1). Second, evidence has accumulated to indicate that the cell surface molecules that mediate cell-cell and cell-ECM interactions (immunoglobulin superfamily, cad-Preparation of this review was supported by NIH Grants HDI 9950 and NS28807

Objective. To assess the effects of human intervertebral disc aggrecan on nerve growth and guidance, using in vitro techniques.Methods. Aggrecan extracted from human lumbar intervertebral discs was incorporated into tissue culture substrata for the culture of the human neuronal cell line, SH-SY5Y, or explants of chick dorsal root ganglia. The effects on nerve growth of different concentrations of aggrecan extracted from the anulus fibrosus and nucleus pulposus, and of these aggrecan preparations following enzymic deglycosylation, were compared.Results. Disc aggrecan inhibited the growth of neurites from SH-SY5Y cells and induced growth cone turning of chick sensory neurites in a concentrationdependent manner. Aggrecan isolated from the anulus fibrosus was more inhibitory than that isolated from the nucleus pulposus, but enzymic pretreatments to reduce the glycosylation of both types of disc aggrecan partially abrogated their inhibitory effects.Conclusion. Nerve growth into degenerate intervertebral discs has been linked with the development of low back pain, but little is known about factors affecting disc innervation. The finding that disc aggrecan inhibits nerve growth in vitro, and that this inhibitory activity depends on aggrecan glycosylation, has important implications for our understanding of mechanisms that may regulate disc innervation in health and disease.

The primary mediators of cell migration during development, wound healing and metastasis, are receptors of the integrin family. In the developing and regenerating nervous system, chondroitin sulfate proteoglycans (CSPGs) inhibit the integrin-dependent migration of neuronal growth cones. Here we report that embryonic sensory neurons cultured on the growth-promoting molecule laminin in combination with the inhibitory CSPG aggrecan rapidly adapt to inhibition. Adaptation is associated with a two- to threefold increase in the levels of RNA and surface protein for two laminin receptors, integrin alpha6beta1 and alpha3beta1, indicating that integrin expression is regulated by aggrecan. Increased integrin expression is associated both with increases in neuronal cell adhesion/outgrowth and with decreases in the ability of aggrecan to inhibit cell adhesion. Directly increasing integrin expression by adenoviral infection is sufficient to eliminate the inhibitory effects of aggrecan, indicating that upregulation of integrin receptors may promote neuronal regeneration in the presence of inhibitory matrix components.

This study examines the mechanisms of spontaneous and induced [Ca2+]i spiking in nerve growth cones and the effect of spikes on growth cone migration. Over a 10-20 min observation period, 29% of DRG growth cones undergo spontaneous and transient elevations in physiological extracellular Ca2+ ((Ca2+)o; 2 mM), whereas 67% of growth cones exposed to 20 mM (Ca2+)o exhibit similar [Ca2+]i spikes. Spontaneous [Ca2+]i spiking was not observed in neuronal cell bodies or nonneuronal cells. Ca2+ influx through non-voltage-gated Ca2+ channels was required for spontaneous [Ca2+]i spikes in growth cones, since removal of (Ca2+)o, or addition of the general Ca2+ channel blockers La3+ or Ni2+, reversibly blocked [Ca2+]i spiking, while blockers of the voltage-gated Ca2+ channels did not. Experiments using agents that influence intracellular Ca2+ stores suggest that Ca2+ stores may buffer and release Ca2+ during growth cone [Ca2+]i spikes. Growth cone migration was immediately and transiently inhibited by [Ca2+]i spikes, but eventually returned to prespike rates.